1. Field of the Invention
The present invention is broadly concerned with test methods or assays designed to determine whether a candidate compound has a significant regulating affect upon cell activities, namely NF-.kappa.B, JNK and apoptosis, so as to facilitate the discovery and/or design of therapeutic agents. The invention is also concerned with novel proteinaceous species useful in such assays, and to methods for regulating cellular NF-.kappa.B, JNK and apoptosis activities for therapeutic purposes.
2. Description of the Prior Art
Nuclear factor-kappa B (NF-.kappa.B) is a critical transcription factor which is required for regulated expression of several genes involved in inflammation and immune response. Five known members of this family have been characterized to date and include c-Rel, p52, RelA and RelB. NF-.kappa.B is present in the cytoplasm of various cells as an inactive complex with an inhibitory protein called I.kappa.B. Stimulation by a number of agents results in the degradation of I.kappa.B and subsequent release of NF-.kappa.B. Once released, NF-.kappa.B is free to migrate to the nucleus and bind to the promoter of specific genes possessing its cognate binding site.
A large number of endogenous and exogenous conditions can lead to the activation of the NF-.kappa.B pathway. These include viral and bacterial infections, environmental toxins, UV irradiation, inflammatory cytokines (TNF.alpha., TNF.beta., IL-1, IL-17, LIF, etc.), growth factors (IL-2, insulin, M-CSF, PDGF, NGF, etc.), immunoreceptor ligands (e.g., CD3 ligand, CD2 ligand, CD28 ligand, CD30 ligand, CD40 ligand, etc.), inflammatory mediators (e.g., Thrombin, angiotensin II, Leukotriene B4), cell adhesion molecules, and several stressful situations (e.g., hypoxia, osmotic shock, hemorrhage, etc.)
Activation of NF-.kappa.B leads to the transcriptional activation of several genes which play a crucial role in both innate and acquired immune responses. Some of the noteworthy genes activated by NF-.kappa.B include those for cytokines and growth factor such as TNF.alpha., TNF.beta., IL-1.beta., IL-2, I, IL-3, IL-6, IL-8, IL-12, GM-CSF, G-CSF, chemokines, cell adhesion molecules, acute phase proteins and transcription factors p53 and c-myc. In addition, several viruses are activated by NF-.kappa.B including the Human immunodeficiency virus 1 (HIV-1) and cytomegalovirus (CMV).
Given the large number of inducers of NF-.kappa.B and equally large number of its target genes, it is not surprising that activation of NF-.kappa.B has been implicated in the pathogenesis of many acute and chronic inflammatory conditions, such as septic shock, rheumatoid arthritis, Crohn's Disease and atherosclerosis. In addition, NF-.kappa.B has a role in oncogenesis and viral transcription regulation, such as in HIV, adenoviruses and papova viruses.
A large number of molecules with immunosuppressive and anti-inflammatory properties have been studied as inhibitors of NF-.kappa.B. These include glucocorticoids and other steroid hormones, cyclosporin A, FK506, rapamycin, salicylates and gold compounds. All these compounds are currently being used for the treatment of several human autoimmune and inflammatory disorders which underscores the clinical and commercial importance of NF-.kappa.B pathway in the control of inflammation and immune response. However, there are two major problems with the above inhibitors. First, the majority of these compounds have broad range of activities so that they will suppress NF-.kappa.B activation by a large number of stimuli. This manifests itself in the form of severe immunosuppression and resulting pre-disposition to opportunistic infections. Second, the majority of these agents have numerous adverse effects associated with the use of these compounds.
Mammalian cells respond to extra cellular stimuli by activation of the MAP kinase family of signaling proteins. Members of the JNK subgroup of MAP kinases are activated in response to diverse extracellular stimuli, including UV irradiation, proinflammatory cytokines and certain mitogens. The JNKs in turn phosphorylate and activate the transcription factor c-Jun, an important component of the transcription activator AP-1. Activation of the JNK pathways has been implicated in a wide variety of responses ranging from cell growth, proliferation, differentiation, cell death, and protection from cell death. As such, this pathway has been implicated in the pathogenesis of human malignancies as well as diseases associated with abnormal cell death. The JNK pathway is activated by several members of the TNFR family. The roll of this pathway and the mediation of cell death by the TNFR family members is still a subject of controversy.
Apoptosis or programmed cell death plays an essential role in the normal growth and development. Abnormalities in this pathway have been linked to the pathogenesis of a number of diseases. For example, failure of cells to undergo apoptosis has been associated with the development of a large number of malignancies, especially low grade lymphomas. Similarly, defects in the apoptosis induced by members of the TNFR family of proteins have been linked to several autoimmune diseases. On the other hand, a large number of diseases are characterized by excessive apoptosis including Alzheimer's disease, AIDS, osteoporosis, ischemic injury, myocardial infarction, and hepatic necrosis. Owing to the central role played by apoptosis in the pathogenesis of several human diseases, the apoptosis pathway and therapies that can modulate this pathway are the focus of extensive research.
Some of the proteins involved in apoptosis have been identified and some interactions among these proteins have been described. However, the mechanisms by which these proteins mediate their activity remains unknown. Given the importance of cell apoptosis and the potential benefits which would flow from effective regulation thereof, considerable research has been undertaken to elucidate the involved proteins and pathway.
Kaposi Sarcoma (KS) is the most common malignancy found in patients with HIV infection. Recent studies have implicated a herpes virus termed Kaposi Sarcoma associated Herpes Virus (KSHV/HHV8) in the pathogenesis of this disease as well as several other malignancies. The mechanism by which KSHV causes malignant transformation is unclear.
Molluscum Contagiosum Virus (MCV) is a benign skin tumor caused by a poxvirus. A hallmark of infection by this virus is an almost complete lack of inflamation in the affected areas which allows the infection to persist for months or even years and to recur after treatment. The mechanism by which MCV blocks the local immune and inflammatory response is unknown.